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The NCI RAS Program

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RAS Pathways & Reagents

NCI researchers have developed unique and powerful reagents at the NCI labs. Below are available pathways and reagents you can purchase.


KRAS Entry & RAS Pathway Clone Collections

Cost: US $38.48 (each requestor must pay shipping expenses) for each collection.

Contact: For additional information on the clone collections, to get more information on the Combinational Cloning Platform, or to order please contact Dominic Esposito, Director, Protein Expression Laboratory, NCI Frederick National Laboratory (FNL) at 301-846-7376 or dom.esposito@nih.gov

For technical questions about the system, please contact Carissa Grose, Protein Expression Laboratory, FNL at 301-360-3427 or grosecl@mail.nih.gov.

Overview

The R3 KRAS Entry Clone Collection #1 is a set of vectors for use with the Gateway Cloning Platform (Life Technologies, Carlsbad, CA) to permit construction of KRAS expression vectors for in vitro and in vivo research. While these clones can be used for standard Gateway cloning, their utility is greatly enhanced with the use of the FNL Combinatorial Cloning Platform (CCP) which allows simplified construction of vectors with different elements.  Some uses of this system might be:

  • Construction of protein expression constructs with various fusion tags
  • Generation of expression constructs with different promoters for in vivo expression
  • Production of clones with fluorescent tags for localization experiments
  • Generation of constructs for making mutant cell lines or transgenic animals
  • Production of vectors for shRNA or miRNA delivery

The advantage of the CCP is in the exquisite specificity of the Multisite Gateway reactions, which permit linkage of multiple elements in a directional fashion, and which involve no additional DNA amplification, thus ensuring the accuracy of the DNA sequence of the final products without the need for additional sequencing.  There is also no need for restriction-based cloning processes which have a high rate of failure and may require optimization depending on the sites available in a given clone.

The KRAS Entry clone collection contains wildtype and mutant KRAS genes in two separate formats.  The “closed” format contains a Kozak translational initiation sequence and ATG start site at the 5’ end, and a stop codon at the 3’ end.  These constructs can be used to make native or aminoterminally tagged constructs using Gateway vectors or the CCP. The “open” format has the same 5’ sequence, but lacks a stop codon and is in frame with the Gateway attB2 site at the 3’ end.  This allows production of C-terminal fusions using Gateway vectors or the CCP.  These clones have been completely sequence validated and functionally tested in Gateway reactions to ensure proper performance.

A large collection of Gateway Multisite vectors tailored for mammalian expression experiments is available through Dr. Esposito. These plasmids combined with the KRAS entry clones allow mix – react – transform construction of plasmids with a variety of promoters (weak, strong, inducible, silencing-resistant) and N- and C-terminal fusions with fluorescent proteins, epitopes, and expression motifs.

Find more information at the PEL Combinatorial Cloning Platform

Gateway Recombinational Cloning

For more information on the Gateway system, please see Hartley JL, Temple GF, and Brasch MA. (2000) “DNA cloning using in vitro site-specific recombination.” Genome Res. 10, 1788-1795.

For more information on the FNLCR Combinatorial Cloning Platform (CCP), please see Wall VA, et. al. (2014) “Combinatorial assembly of clone libraries using site-specific recombination.” Meth. Mol. Biol. 1116, 193-208.


KRAS-4b wt and Mutant Expression Plasmids Clone Collection 1

KRAS Entry Clone Collection 1 (Wild-type KRAS and 10 point mutations / Open and close formats / Plasmid DNA and glycerol stocks of E. coli DH10B harboring each plasmid)

The NCI RAS Program collection of KRAS entry clones comprises 11 KRAS-4b genes, each in the closed (i.e., with a stop codon) and open (no stop codon, for C-terminal fusions) configuration. Both DNA and frozen E. coli cells are provided.  Miniprep plasmid DNA for all Entry clones is provided, and concentrations are noted on the vials.  In addition, glycerol stocks of E. coli DH10B cells containing the plasmids are provided. Gateway Entry clones are in the pDonr255 backbone and should be propagated with 50 ug/ml spectinomycin to ensure plasmid stability.  

The NCI KRAS entry clone collection includes wild type KRAS-4b and the following mutants:

  • G12C
  • G12D
  • G12V
  • G13D
  • Q61L
  • Q61R
  • S181A
  • S181D
  • S181E
  • C185S

All clones are in a standard attL1-attL2 Gateway Entry vector (pDonr255) and contain a 5’ Kozak translation initiation sequence prior to the ATG start codon.

Clone IdentifierGeneC-terminus

R750-E01

Hs.KRAS4b

closed

R750-E02

Hs.KRAS4b

open

R750-E03

Hs.KRAS4b G12C

closed

R750-E04

Hs.KRAS4b G12C

open

R750-E05

Hs.KRAS4b G12D

closed

R750-E06

Hs.KRAS4b G12D

open

R750-E07

Hs.KRAS4b G12V

closed

R750-E08

Hs.KRAS4b G12V

open

R750-E09

Hs.KRAS4b G13D

closed

R750-E10

Hs.KRAS4b G13D

open

R750-E11

Hs.KRAS4b Q61L

closed

R750-E12

Hs.KRAS4b Q61L

open

R750-E13

Hs.KRAS4b Q61R

closed

R750-E14

Hs.KRAS4b Q61R

open

R750-E15

Hs.KRAS4b S181A

closed

R750-E16

Hs.KRAS4b S181A

open

R750-E17

Hs.KRAS4b S181D

closed

R750-E18

Hs.KRAS4b S181D

open

R750-E19

Hs.KRAS4b S181E

closed

R750-E20

Hs.KRAS4b S181E

open

R750-E21

Hs.KRAS4b C185S

closed

R750-E22

Hs.KRAS4b C185S

open

 


Expression Plasmids for 15 RAS Pathway Genes

RAS Entry Clone Collection 1: 15 RAS pathway genes; Open and closed formats; Plasmid DNA and glycerol stocks of E. coli DH10B harboring each plasmid

Researchers within the NCI RAS Program has constructed a set of Gateway-compatible entry clones for the following 15 RAS pathway genes. All clones are available with and without stop codons (the latter configuration allows for C-terminal fusions). Miniprep plasmid DNA for all Entry clones is provided, and concentrations are noted on the vials.  In addition, glycerol stocks of E. coli DH10B cells containing the plasmids are provided. Gateway Entry clones are in the pDonr255 backbone and should be propagated with 50 ug/ml spectinomycin to ensure plasmid stability.  

Both DNA and frozen E. coli cells are provided. The genes include:

  • SOS1
  • SOS2
  • RASGRP1
  • RASGRP2
  • RASGRP3
  • RASGEF1A
  • KNDC1
  • RASA1
  • SYNGAP1
  • RASAL1
  • RASA4
  • BRAF 
  • RAF1
  • ARAF
  • PDE6D

All clones are in a standard attL1-attL2 Gateway Entry vector (pDonr255) and contain a 5’ Kozak translation initiation sequence prior to the ATG start codon. Genbank RefSeq identifiers are listed below to identify the specific isoforms of the genes in these clones.

Clone IdentifierGeneGenBank RefSeq IdentifierC-terminus
R702-E01Hs.SOS1

NM_005633.3

closed

R702-E02Hs.SOS1

NM_005633.3

open

R702-E03Hs.SOS2

NM_006939.2

closed

R702-E04Hs.SOS2

NM_006939.2

open

R702-E05Hs.RASGRP1

NM_005739.3

closed

R702-E06Hs.RASGRP1

NM_005739.3

open

R702-E07Hs.RASGRP2

NM_153819.1

closed

R702-E08Hs.RASGRP2

NM_153819.1

open

R702-E09Hs.RASGRP3

NM_001139488.1

closed

R702-E10Hs.RASGRP3

NM_001139488.1

open

R702-E11Hs.RASGEF1A

NM_145313.2

closed

R702-E12Hs.RASGEF1A

NM_145313.2

open

R702-E13Hs.KNDC1

NM_152643.6

closed

R702-E14Hs.KNDC1

NM_152643.6

open

R702-E15Hs.RASA1

NM_002890.2

closed

R702-E16Hs.RASA1

NM_002890.2

open

R702-E17Hs.SYNGAP1

NM_006772.2

closed

R702-E18Hs.SYNGAP1

NM_006772.2

open

R702-E19Hs.RASAL1

NM_001193520.1

closed

R702-E20Hs.RASAL1

NM_001193520.1

open

R702-E21Hs.RASA4

NM_006989.5

closed

R702-E22Hs.RASA4

NM_006989.5

open

R702-E23Hs.BRAF

NM_004333.4

closed

R702-E24Hs.BRAF

NM_004333.4

open

R702-E25Hs.RAF1

NM_002880.3

closed

R702-E26Hs.RAF1

NM_002880.3

open

R702-E27Hs.ARAF

NM_001654.4

closed

R702-E28Hs.ARAF

NM_001654.4

open

R702-E29Hs.PDE6D

NM_002601.3

closed

R702-E30Hs.PDE6D

NM_002601.3

open